Cell polarization and directed migration are fundamental processes required for human cells to mediate immune responses, wound healing, angiogenesis, development, and cancer metastasis. While much work has been done to identify and characterize regulatory proteins involved in these processes, the large number of critical proteins and second messengers has left the questions open of how cells induce polarization, drive migration and trigger chemotaxis towards a source of chemoattractant. The goal of the proposed work is to answer these questions by employing a microscopy based quantitative analysis of the regulatory control systems in endothelial cells and neutrophils. Understanding how these regulatory systems are build will likely reveal critical control points that have potentil as therapeutic targets. Our specific strategy is based on cell lines that we developed expressing FRET- and translocation- based fluorescent biosensors for three different core second messengers, PIP3, Ca2+ and diacylgycerol as well as for four core small GTPases, Ras, RhoA, Rac and Cdc42. We have also developed rapid perturbation strategies, based on synthetic expressed DNA constructs that allow us to use small molecules to rapidly activate each of these core regulators of polarization and identify and characterize the relevant local feedback loops in the two cell models. This already led to the identification of important proteins that connect these core regulators to each other. Furthermore, by being able to track cells and monitor local signaling activities as they migrate and turn towards sources of chemoattractant, we have been able to identify an unexpected central role of Cdc42 in the polarization and directed migration of both cells. The fundamental nature of our experiments on polarization and directed cell migration will make our studies relevant to understand these processes in a wide range of cell types and organisms. Our studies will also provide important tools for other researchers to use for studies of polarization and migration.